Precursor tri-specific antibody constructs and methods of use thereof

Abstract

Precursor tri-specific antibody constructs comprising at least one tumor associated antigen binding domain, a T-cell binding domain, and a regulatory domain having enhanced half-life, are described herein. Further, methods of producing the precursor tri-specific antibody constructs are disclosed. Pharmaceutical compositions comprising the precursor constructs and their uses for treating tumors are also disclosed.

1 . A precursor tri-specific antibody construct, comprising:
 (a) a first binding domain that binds to a first tumor associated antigen (TAA);   (b) a second binding domain that binds to a second TAA;   (c) a third binding domain that binds to an extracellular epitope of human CD3ε; and   (d) a regulatory domain, said regulatory domain comprising either
 (i) a first and a second sub-regulatory domain, said first sub-regulatory domain comprising a first protease cleavage domain and a half-life prolonging (HLP) domain, and said second sub-regulatory domain comprising a second protease cleavage domain and a CAP component that reduces the ability of the third binding domain to bind the extracellular epitope of human CD3ε; or 
 (ii) a single regulatory domain comprising a protease cleavage domain, a half-life prolonging (HLP) domain, and a CAP component that reduces the ability of the third binding domain to bind the extracellular epitope of human CD3ε. 
   
     
     
         2 . The precursor tri-specific antibody construct of  claim 1 , wherein said first binding domain and said second binding domain bind to the same TAA or bind to different TAAs. 
     
     
         3 . The precursor tri-specific antibody construct of  claim 1 , wherein said first TAA, or said second TAA, or both said first TAA and said second TAA are selected from the group consisting of an extracellular epitope of a tumor-cell-surface antigen, a tumor micro-environment antigen, a stromal antigen in the tumor micro-environment (TME), an angiogenic antigen in the TME, an antigen on a blood vessel in a TME, a cytokine antigen in a TME, and any combination thereof. 
     
     
         4 . The precursor tri-specific antibody construct of  claim 3 , wherein said TAA bound by said first binding domain or said second binding domain or both is selected from the group consisting of 5T4, ROR1, EGFR, and PSMA. 
     
     
         5 . The precursor tri-specific antibody construct of  claim 4 , wherein
 (a) when said TAA is 5T4, the first binding domain or the second binding domain or both comprise the amino acid sequence set forth in any one of SEQ ID NOs: 172 and 174, or a combination thereof;   (b) when said TAA is ROR1, the first binding domain or the second binding domain or both comprise the amino acid sequence set forth in any one of SEQ ID NOs: 156 and 166, or a combination thereof;   (c) when said TAA is EGFR, the first binding domain or the second binding domain or both comprise the amino acid sequence set forth in any one of SEQ ID NOs: 34, 37, or a combination thereof; and   (d) when said TAA is PSMA, the first binding domain or the second binding domain or both comprise the amino acid sequence set forth in any one of SEQ ID NOs: 168 and 170, or a combination thereof.   
     
     
         6 . The precursor tri-specific antibody construct of  claim 3 , wherein said tumor micro-environment antigen is selected from the group consisting of KIR, LILR, and TIGIT. 
     
     
         7 . The precursor tri-specific antibody construct of  claim 3 , wherein said stromal antigen in the tumor micro-environment is selected from the group consisting of fibroblast activation protein (FAP), alpha smooth muscle actin (αSMA), PDGFRα, Integrin α11β1(ITGA11)VEGF, Tenascin-C, periostin, fibroblast specific protein 1 (S10A4, FSP1), desmin, vimentin, paladin, urokinase-type plasminogen activator receptor associated protein (UPARAP), galectin-3, podoplanin, platelet, CCL2, and CXCL12. 
     
     
         8 . The precursor tri-specific antibody construct of  claim 3 , wherein said angiogenic antigen in the tumor micro-environment is selected from the group consisting of bFGF, INF, and VEGF. 
     
     
         9 . The precursor tri-specific antibody construct of  claim 3 , wherein said antigen on the surface of a blood vessel in the tumor micro-environment is selected from the group consisting of CD31, CD105, CD146, and CD144. 
     
     
         10 . The precursor tri-specific antibody construct of  claim 3 , wherein said cytokine antigen is selected from the group consisting of TNF-alpha, IL-6, TGF-beta, IL-10, IL-8, IL-17, IL-21, INF, and VEG. 
     
     
         11 . The precursor tri-specific antibody construct of  claim 1 , wherein the HLP domain comprises a human serum albumin (HSA) polypeptide. 
     
     
         12 . The precursor tri-specific antibody construct of  claim 1 , wherein the CAP component comprises an amino acid sequence of the extracellular epitope of human CD3ε. 
     
     
         13 . The precursor tri-specific antibody construct of  claim 1 , wherein the CAP component comprises an amino acid sequence as set forth in SEQ ID NO: 5, or a homolog thereof. 
     
     
         14 . The precursor tri-specific antibody construct of  claim 1 , wherein the first binding domain, the second binding domain, or both, each comprises a single chain variable fragment (scFv). 
     
     
         15 . The precursor tri-specific antibody construct of  claim 1 , wherein the third binding domain comprises a Fab antigen binding fragment. 
     
     
         16 . The precursor tri-specific antibody construct of  claim 1 , wherein the protease cleavage domain in the first and second sub-regulatory domains are cleaved by the same protease or different proteases. 
     
     
         17 . The precursor tri-specific antibody construct of  claim 1 , wherein one or both of the first and second protease cleavage domain comprises a protease-cleavable amino acid sequence cleavable by a serine protease, a cysteine protease, an aspartate protease, a matrix metalloprotease (MMP), or is a combination substrate cleaved by one or more of MMP2/9, uPA, matriptase and legumain, or any combination thereof. 
     
     
         18 . A pharmaceutical composition comprising the precursor tri-specific antibody construct of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         19 . A nucleic acid construct comprising one or more nucleic acid sequences that encode the precursor tri-specific antibody construct of  claim 1 . 
     
     
         20 . An expression vector comprising the nucleic acid construct of  claim 19 . 
     
     
         21 . An isolated host cell comprising the expression vector of  claim 20 . 
     
     
         22 . A method of treating, preventing, inhibiting the growth of, delaying disease progression, reducing tumor load, or reducing the incidence of a cancer or a tumor, or any combination thereof, in a subject in need of such treatment, comprising a step of administering to the subject the pharmaceutical composition of  claim 18 , wherein the method treats, prevents, inhibits the growth of, delays the disease progression, reduces the tumor load, or reduces the incidence of the cancer or a tumor in the subject. 
     
     
         23 . The method of  claim 22 , wherein the cancer or tumor comprises a solid tumor or non-solid tumor, or wherein the cancer or tumor comprises a metastasis of a cancer or tumor.